Inhibition of ubiquitin-activating enzyme protects against organ injury after intestinal ischemia-reperfusion.

Intestinal ischemia-reperfusion (I/R) occurs in various clinical settings, such as transplantation, acute mesenteric arterial occlusion, trauma, and shock. I/R injury causes severe systemic inflammation, leading to multiple organ dysfunction associated with high mortality. The ubiquitin proteasome pathway has been indicated in the regulation of inflammation, particularly through the NF-κB signaling pathway. PYR-41 is a small molecular compound that selectively inhibits ubiquitin-activating enzyme E1. A mouse model of intestinal I/R injury by clamping the superior mesenteric artery for 45 min was performed to evaluate the effect of PYR-41 treatment on organ injury and inflammation. PYR-41 was administered intravenously at the beginning of reperfusion. Blood and organ tissues were harvested at 4 h after reperfusion. PYR-41 treatment improved the morphological structure of gut and lung after I/R, as judged by hematoxylin and eosin staining. It also reduced the number of apoptotic terminal deoxynucleotidyl transferase dUTP nick end-labeling-positive cells and caspase-3 activity in the organs. PYR-41 treatment decreased the expression of proinflammatory cytokines IL-6 and IL-1ß as well as chemokines keratinocyte chemoattractant and macrophage inflammatory protein-2 in the gut and lung, which leads to inhibition of neutrophils infiltrating into these organs. The serum levels of IL-6, aspartate aminotransferase, and lactate dehydrogenase were reduced by the treatment. The IκB degradation in the gut increased after I/R was inhibited by PYR-41 treatment. Thus, ubiquitination may be a potential therapeutic target for treating patients suffering from intestinal I/R. NEW & NOTEWORTHY Excessive inflammation contributes to organ injury from intestinal ischemia-reperfusion (I/R) in many clinical conditions. NF-κB signaling is very important in regulating inflammatory response. In an experimental model of gut I/R injury, we demonstrate that administration of a pharmacological inhibitor of ubiquitination process attenuates NF-κB activation, leading to reduction of inflammation, tissue damage, and apoptosis in the gut and lungs. Therefore, ubiquitination process may serve as a therapeutic target for treating patients with intestinal I/R injury.

AMPK inhibitor compound C suppresses cell proliferation by induction of apoptosis and autophagy in human colorectal cancer cells.

BACKGROUND AND OBJECTIVES: AMP-activated protein kinase (AMPK) is a main regulator of energy metabolism through the inhibition of biosynthetic pathways and enhancement of ATP-generating pathways. However, targeting AMPK as anti-tumor therapy remains controversial. In this study, we examined the effect of compound C, a small molecule inhibitor of AMPK, on the proliferation of several human colorectal cancer cell lines with diverse characteristics. METHODS: Four human colorectal cancer cell lines (HCT116, DLD-1, SW480, and KM12C) were treated with compound C. Cell viability was determined by MTS assay. Cell cycle prolife was analyzed by flow cytometry. Acidic vesicular organelles were detected by acridine orange staining. Protein levels were measured by western blotting. RESULTS: Compound C inhibited the growth of four cell lines in a dose-dependent manner and caused G(2) /M arrest. Compound C increased sub-G(1) cell population and induced chromatin condensation and cleavage of PARP in HCT116 and KM12C cells, while it induced acidic vesicular formation and conversion of LC3-I to autophagosome-associated LC3-II in DLD-1 and SW480 cells. Survivin, an anti-apoptotic protein, was down-regulated in all cell lines treated with compound C. CONCLUSIONS: Compound C induces apoptotic or autophagic death in colorectal cancer cells and the preferred death mode is cell type-dependent.